The most widely utilized RIA for inhibin in humans is flawed in that it recognizes free beta-inhibin proteins in addition to dimeric, bioactive inhibin. Thus, most of the data concerning the role of inhibin in normal human reproduction as well as in disease states is highly suspect. The solution to this methodological quagmire is to develop an immunoassay that is specific for dimeric, bioactive inhibin and is sufficiently sensitive for quantification of inhibin in human serum samples. Specific Aim #1 of this proposal concerns development and validation of such an assay, consisting of a two-site, alpha, beta sandwich Elisa. Toward this end, we will generate an extensive panel of monoclonal and polyclonal antibodies, characterize them as to binding specificity and affinity, and assemble the most promising antibodies into assays for validation. We will then reexamine inhibin physiology in human males and females and establish a normative database. These data will be compared to inhibin levels in a wide array of infertile patients (Specific Aim #2) to determine inhibin's role in disease states involving gonadal failure. In Specific Aim #3, the biochemistry of inhibin synthesis and secretion will be investigated in normal and infertile patients and related to RIA levels of this hormone. Since the forms of inhibin that are naturally produced and secreted in humans have not yet been identified, they will be purified from human follicular fluid by chromatography and characterized by electrophoresis and Western blot analysis. Dimeric inhibin levels will be compared quantitatively to free alpha-inhibin subunit as an index of abnormal production or secretion of inhibin in infertile patients. Lastly, in Specific Aim #4, we will investigate extragonadal sources of inhibin, such as the adrenal, and characterize their ramification to use of inhibin concentrations as an index of gonadal function as well as a correlate of adrenal function through clinical stages of normal and delayed puberty. The results of this research will provide, for the first time, an accurate understanding of inhibin physiology in normal humans, as well as inhibin's role in human infertility. In addition, it will provide technology that could be utilized by other investigators in related aspects of inhibin physiology and biochemistry.